Multifunctional Milk-Derived Small Extracellular Vesicles and Their Biomedical Applications.

In recent years, small extracellular vesicles (sEVs) have been regarded as the next generation of novel delivery systems after lipid nanoparticles because of their advantages and huge prospects in drug delivery. Studies have shown that sEVs are abundant in milk and therefore can be a large and economical source of sEVs. Natural milk-derived small extracellular vesicles (msEVs) have important functions such as immune regulation, anti-bacterial infection, anti-oxidative, etc., and play a beneficial role in human health at multiple levels, including intestinal health, bone/muscle metabolism, and microbiota regulation. In addition, because they can pass the gastrointestinal barrier and have low immunogenicity, good biocompatibility, and stability, msEVs are considered a crucial oral drug delivery vehicle. Moreover, msEVs can be further engineered for targeted delivery to prolong the circulation time or enhance local drug concentrations. However, msEVs separation and purification, complex contents, and quality control hinder their application in drug delivery. This paper provides a comprehensive review of the biogenesis and characteristics, isolation and purification, composition, loading methods, and function of msEVs, based on which their applications in biomedical fields are further explored.

Exosomal ncRNAs: The pivotal players in diabetic wound healing.

Diabetes is the most prevalent metabolic disease in the world today. In addition to elevated blood glucose, it also causes serious complications, which has a significant effect on the quality of life of patients. Diabetic trauma is one of complications as a result of the interaction of diabetic neuropathy, peripheral vascular disease, infection, trauma, and other factors. Diabetic trauma usually leads to poor healing of the trauma and even to severe foot ulcers, wound gangrene, and even amputation, causing serious psychological, physical, and financial burdens to diabetic patients. Non-coding RNAs (ncRNAs) carried by exosomes have been demonstrated to be relevant to the development and treatment of diabetes and its complications. Exosomes act as vehicle, which contain nucleic acids such as mRNA and microRNA (miRNA), and play a role in the intercellular communication and the exchange of substances between cells. Because exosomes are derived from cells, there are several advantages over synthetic nanoparticle including good biocompatibility and low immunogenicity. Exosomal ncRNAs could serve as markers for the clinical diagnosis of diabetes and could also be employed to accelerate diabetic wound healing via the regulation of the immune response and modulation of cell function. ncRNAs in exosomes can be employed to promote diabetic wound healing by regulating inflammation and accelerating re-vascularization, re-epithelialization, and extracellular matrix remodeling. Herein, exosomes in terms of ncRNA (miRNA, lncRNA, and circRNA) to accelerate diabetic wounds healing were summarized, and we discussed the challenge of the loading strategy of ncRNA into exosomes.

N-glycoproteomic profiling revealing novel coronavirus therapeutic targets potentially involved in Cepharanthine's intervention.

The Coronavirus disease 2019 (COVID-19) has posed a serious threat to global health and the world economy. Antiviral therapies targeting coronavirus are urgently required. The Cepharanthine (CEP) is a traditional Chinese herbal extract. Our previous research revealed that CEP has a very potent anti-coronavirus effect, but its mechanism of action was not fully understood. To investigate the effect of novel coronavirus on protein glycosylation in infected cells and to further investigate the mechanism of action of CEP against coronavirus, a cellular model using coronavirus GX_P2V infection of Vero E6 cells was established. The effect of coronavirus GX_P2V on host cell protein glycosylation was investigated by N-glycoproteomic analysis, and the antagonistic effect of CEP on the abnormal protein glycosylation caused by coronavirus was analyzed. The results showed that GX_P2V could cause abnormal changes in protein glycosylation levels in host cells, while CEP could partially antagonize the abnormal protein glycosylation caused by GX_P2V. In addition, we also found that CEP could regulate the glycosylation level of coronavirus S protein. In conclusion, this article provides important ideas about the infection mechanism of novel coronaviruses, providing evidence for CEP as a promising therapeutic option for coronavirus infection.

Advances in Circular RNA and Its Applications.

Circular RNA (circRNA) is a novel endogenous non-coding RNA (ncRNA) that, like microRNA (miRNA), is a rapidly emerging RNA research topic. CircRNA, unlike traditional linear RNAs (which have 5' and 3' ends), has a closed-loop structure that is unaffected by RNA exonucleases. Thus, circRNA has sustained expression and is less sensitive to degradation. Since circRNAs have many miRNAs binding sites, eliminating their repressive effects on their target genes can strongly enhance their expression. CircRNAs serve an important regulatory role in disease onset and progression via specific circRNA-miRNA interactions. We summarized the current progress in elucidating mechanisms and biogenesis of circRNAs in this review. In particular, circRNAs can function mainly as miRNA sponges, regulating host gene expression and protein transportation. Finally, we discussed the application prospects and significant challenges for the development of circRNA-based therapeutics.

Biomimetic Exosomes: A New Generation of Drug Delivery System.

Most of the naked drugs, including small molecules, inorganic agents, and biomacromolecule agents, cannot be used directly for disease treatment because of their poor stability and undesirable pharmacokinetic behavior. Their shortcomings might seriously affect the exertion of their therapeutic effects. Recently, a variety of exogenous and endogenous nanomaterials have been developed as carriers for drug delivery. Among them, exosomes have attracted great attention due to their excellent biocompatibility, low immunogenicity, low toxicity, and ability to overcome biological barriers. However, exosomes used as drug delivery carriers have significant challenges, such as low yields, complex contents, and poor homogeneity, which limit their application. Engineered exosomes or biomimetic exosomes have been fabricated through a variety of approaches to tackle these drawbacks. We summarized recent advances in biomimetic exosomes over the past decades and addressed the opportunities and challenges of the next-generation drug delivery system.

Host Cyanobacteria Killing by Novel Lytic Cyanophage YongM: A Protein Profiling Analysis.

Cyanobacteria are autotrophic prokaryotes that can proliferate robustly in eutrophic waters through photosynthesis. This can lead to outbreaks of lake "water blooms", which result in water quality reduction and environmental pollution that seriously affect fisheries and aquaculture. The use of cyanophages to control the growth of cyanobacteria is an important strategy to tackle annual cyanobacterial blooms. YongM is a novel lytic cyanophage with a broad host spectrum and high efficiency in killing its host, cyanobacteria FACHB-596. However, changes in cyanophage protein profile during infestation and killing of the host remains unknown. To characterize the proteins and its regulation networks involved in the killing of host cyanobacteria by YongM and evaluate whether this strain YongM could be used as a chassis for further engineering to be a powerful tool in dealing with cyanobacterial blooms, we herein applied 4D label-free high-throughput quantitative proteomics to analyze differentially expressed proteins (DEPs) involved in cyanobacteria host response infected 1 and 8 h with YongM cyanophage. Metabolic pathways, such as photosynthesis, photosynthesis-antennal protein, oxidative phosphorylation, ribosome, carbon fixation, and glycolysis/glycol-isomerization were significantly altered in the infested host, whereas DEPs were associated with the metabolic processes of photosynthesis, precursor metabolites, energy production, and organic nitrogen compounds. Among these DEPs, key proteins involved in YongM-host interaction may be photosystem I P700 chlorophyll-a apolipoprotein, carbon dioxide concentration mechanism protein, cytochrome B, and some YongM infection lysis-related enzymes. Our results provide comprehensive information of protein profiles during the invasion and killing of host cyanobacteria by its cyanophage, which may shed light on future design and manipulation of artificial cyanophages against water blooms.

A Novel Wide-Range Freshwater Cyanophage MinS1 Infecting the Harmful Cyanobacterium Microcystis aeruginosa.

Microcystis aeruginosa, as one of the major players in algal bloom, produces microcystins, which are strongly hepatotoxic, endangering human health and damaging the ecological environment. Biological control of the overgrowth of Microcystis with cyanophage has been proposed to be a promising solution for algal bloom. In this study, a novel strain of Microcystis cyanophage, MinS1, was isolated. MinS1 contains an icosahedral head approximately 54 nm in diameter and a 260 nm-long non-contractile tail. The phage genome consists of a linear, double-stranded 49,966 bp DNA molecule, which shares very low homology with known phages in the NCBI database (only 1% of the genome showed weak homology with known phages when analyzed by megablast). The phage contains 75 ORFs, of which 23 ORFs were predicted to code for proteins of known function, 39 ORFs were predicted to code for proteins of unknown function, and 13 ORFs showed no similarity to any protein sequences. Transmission electron microscopy and phylogenetic analysis showed that MinS1 belongs to the family Siphoviridae. Various experiments confirmed that the phage could infect several different orders of cyanobacteria, including Chroococcales, Nostocales, Oscillatoriales, Hormogonales, and Synechococcales, indicating that it has a very broad host range. In addition, MinS1 has no known antibiotic tolerance genes, virulence genes, and tRNAs, and it is tolerant to temperature, pH, UV, and salinity, suggesting that MinS1 has good potential for application as a biological control agent against cyanobacterial blooms. This study expands the diversity and knowledge of cyanophages, and it provides useful information for the development of novel prevention and control measures against cyanobacterial blooms.

Advances in Synthetic Biology and Biosafety Governance.

Tremendous advances in the field of synthetic biology have been witnessed in multiple areas including life sciences, industrial development, and environmental bio-remediation. However, due to the limitations of human understanding in the code of life, any possible intended or unintended uses of synthetic biology, and other unknown reasons, the development and application of this technology has raised concerns over biosafety, biosecurity, and even cyberbiosecurity that they may expose public health and the environment to unknown hazards. Over the past decades, some countries in Europe, America, and Asia have enacted laws and regulations to control the application of synthetic biology techniques in basic and applied research and this has resulted in some benefits. The outbreak of the COVID-19 caused by novel coronavirus SARS-CoV-2 and various speculations about the origin of this virus have attracted more attention on bio-risk concerns of synthetic biology because of its potential power and uncertainty in the synthesis and engineering of living organisms. Therefore, it is crucial to scrutinize the control measures put in place to ensure appropriate use, promote the development of synthetic biology, and strengthen the governance of pathogen-related research, although the true origin of coronavirus remains hotly debated and unresolved. This article reviews the recent progress made in the field of synthetic biology and combs laws and regulations in governing bio-risk issues. We emphasize the urgent need for legislative and regulatory constraints and oversight to address the biological risks of synthetic biology.

Transcriptome analysis of cepharanthine against a SARS-CoV-2-related coronavirus.

Antiviral therapies targeting the pandemic coronavirus disease 2019 (COVID-19) are urgently required. We studied an already-approved botanical drug cepharanthine (CEP) in a cell culture model of GX_P2V, a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-related virus. RNA-sequencing results showed the virus perturbed the expression of multiple genes including those associated with cellular stress responses such as endoplasmic reticulum (ER) stress and heat shock factor 1 (HSF1)-mediated heat shock response, of which heat shock response-related genes and pathways were at the core. CEP was potent to reverse most dysregulated genes and pathways in infected cells including ER stress/unfolded protein response and HSF1-mediated heat shock response. Additionally, single-cell transcriptomes also confirmed that genes of cellular stress responses and autophagy pathways were enriched in several peripheral blood mononuclear cells populations from COVID-19 patients. In summary, this study uncovered the transcriptome of a SARS-CoV-2-related coronavirus infection model and anti-viral activities of CEP, providing evidence for CEP as a promising therapeutic option for SARS-CoV-2 infection.

The establishment of humanized IL-4/IL-4RA mouse model by gene editing and efficacy evaluation.

Asthma is a common respiratory immune disease in children and adults, and interleukin-4 (IL-4) is one of the key factors for the onset of asthma. Therefore, targeting human IL-4 and IL-4 receptor alpha (IL-4RA) has become one of the strategies for targeted therapy of cytokines. Herein, we established an animal model of asthmatic airway inflammation using double humanized IL-4/IL-4RA (hIL-4/hIL-4RA) mice, where human IL-4 and IL-4RA replaced their murine counterparts, respectively. We successfully identified the phenotype by Southern blotting, ELISA, and flow cytometry. The hIL-4/hIL-4RA mice induced by ovalbumin (OVA) exhibited several important features of asthma, such as inflammatory cell infiltration, IgE release, goblet cell hyperplasia, and Th2 cytokine secretion. Furthermore, treatment of these humanized mice with anti-human IL-4RA antibodies significantly inhibited level of these pathological indicators. Thus, hIL-4/hIL-4RA mice provide a validated preclinical mouse model to interrogate new therapeutic agents targeting this specific cytokine pathway in asthma.

Engineering FKBP-Based Destabilizing Domains to Build Sophisticated Protein Regulation Systems.

Targeting protein stability with small molecules has emerged as an effective tool to control protein abundance in a fast, scalable and reversible manner. The technique involves tagging a protein of interest (POI) with a destabilizing domain (DD) specifically controlled by a small molecule. The successful construction of such fusion proteins may, however, be limited by functional interference of the DD epitope with electrostatic interactions required for full biological function of proteins. Another drawback of this approach is the remaining endogenous protein. Here, we combined the Cre-LoxP system with an advanced DD and generated a protein regulation system in which the loss of an endogenous protein, in our case the tumor suppressor PTEN, can be coupled directly with a conditionally fine-tunable DD-PTEN. This new system will consolidate and extend the use of DD-technology to control protein function precisely in living cells and animal models.

Ubiquitin E3 ligase Nedd4-1 acts as a downstream target of PI3K/PTEN-mTORC1 signaling to promote neurite growth.

Protein ubiquitination is a core regulatory determinant of neural development. Previous studies have indicated that the Nedd4-family E3 ubiquitin ligases Nedd4-1 and Nedd4-2 may ubiquitinate phosphatase and tensin homolog (PTEN) and thereby regulate axonal growth in neurons. Using conditional knockout mice, we show here that Nedd4-1 and Nedd4-2 are indeed required for axonal growth in murine central nervous system neurons. However, in contrast to previously published data, we demonstrate that PTEN is not a substrate of Nedd4-1 and Nedd4-2, and that aberrant PTEN ubiquitination is not involved in the impaired axon growth upon deletion of Nedd4-1 and Nedd4-2. Rather, PTEN limits Nedd4-1 protein levels by modulating the activity of mTORC1, a protein complex that controls protein synthesis and cell growth. Our data demonstrate that Nedd4-family E3 ligases promote axonal growth and branching in the developing mammalian brain, where PTEN is not a relevant substrate. Instead, PTEN controls neurite growth by regulating Nedd4-1 expression.

Orthogonal gene expression in Escherichia coli.

Here, we describe a route orthogonal gene expression which combines orthogonal transcription and translation using library-based selections. We show how orthogonal gene expression can be used to create a minimal orthogonal ribosome and describe how to create orthogonal transcription-translation feed forward loops that introduce tailored information processing delays into gene expression.

Genetically encoded photocontrol of protein localization in mammalian cells.

Precise photochemical control of protein function can be achieved through the site-specific introduction of caging groups. Chemical and enzymatic methods, including in vitro translation and chemical ligation, have been used to photocage proteins in vitro. These methods have been extended to allow the introduction of caged proteins into cells by permeabilization or microinjection, but cellular delivery remains challenging. Since lysine residues are key determinants for nuclear localization sequences, the target of key post-translational modifications (including ubiquitination, methylation, and acetylation), and key residues in many important enzyme active sites, we were interested in photocaging lysine to control protein localization, post-translational modification, and enzymatic activity. Photochemical control of these important functions mediated by lysine residues in proteins has not previously been demonstrated in living cells. Here we synthesized 1 and evolved a pyrrolysyl-tRNA synthetase/tRNA pair to genetically encode the incorporation of this amino acid in response to an amber codon in mammalian cells. To exemplify the utility of this amino acid, we caged the nuclear localization sequences (NLSs) of nucleoplasmin and the tumor suppressor p53 in human cells, thus mislocalizing the proteins in the cytosol. We triggered protein nuclear import with a pulse of light, allowing us to directly quantify the kinetics of nuclear import.

Regulation of cellular metabolism by protein lysine acetylation.

Protein lysine acetylation has emerged as a key posttranslational modification in cellular regulation, in particular through the modification of histones and nuclear transcription regulators. We show that lysine acetylation is a prevalent modification in enzymes that catalyze intermediate metabolism. Virtually every enzyme in glycolysis, gluconeogenesis, the tricarboxylic acid (TCA) cycle, the urea cycle, fatty acid metabolism, and glycogen metabolism was found to be acetylated in human liver tissue. The concentration of metabolic fuels, such as glucose, amino acids, and fatty acids, influenced the acetylation status of metabolic enzymes. Acetylation activated enoyl-coenzyme A hydratase/3-hydroxyacyl-coenzyme A dehydrogenase in fatty acid oxidation and malate dehydrogenase in the TCA cycle, inhibited argininosuccinate lyase in the urea cycle, and destabilized phosphoenolpyruvate carboxykinase in gluconeogenesis. Our study reveals that acetylation plays a major role in metabolic regulation.

Synthesis of orthogonal transcription-translation networks.

Orthogonal, parallel and independent, systems are one key foundation for synthetic biology. The synthesis of orthogonal systems that are uncoupled from evolutionary constraints, and selectively abstracted from cellular regulation, is an emerging approach to making biology more amenable to engineering. Here, we combine orthogonal transcription by T7 RNA polymerase and translation by orthogonal ribosomes (O-ribosomes), creating an orthogonal gene expression pathway in Escherichia coli. We design and implement compact, orthogonal gene expression networks. In particular we focus on creating transcription-translation feed-forward loops (FFLs). The transcription-translation FFLs reported cannot be created by using the cells' gene expression machinery and introduce information-processing delays on the order of hours into gene expression. We refactor the rRNA operon, uncoupling the synthesis of the orthogonal 16S rRNA for the O-ribosome from the synthesis and processing of the rest of the rRNA operon, thereby defining a minimal module that can be added to the cell for O-ribosome production. The minimal O-ribosome permits the rational alteration of the delay in an orthogonal gene expression FFL. Overall this work demonstrates that system-level dynamic properties are amenable to rational manipulation and design in orthogonal systems. In the future this system may be further evolved and tuned to provide a spectrum of tailored dynamics in gene expression and investigate the effects of delays in cellular decision-making processes.

P70 S6 kinase mediates tau phosphorylation and synthesis.

Currently, we found that the 70-kDa p70 S6 kinase (p70S6K) directly phosphorylates tau at S262, S214, and T212 sites in vitro. By immunoprecipitation, p-p70S6K (T421/S424) showed a close association with p-tau (S262 and S396/404). Zinc-induced p70S6K activation could only upregulate translation of total S6 and tau but not global proteins in SH-SY5Y cells. The requirement of p70S6K activation was confirmed in the SH-SY5Y cells that overexpress wild-type htau40. Level of p-p70S6K (T421/S424) was only significantly correlated with p-tau at S262, S214, and T212, but not T212/S214, in Alzheimer's disease (AD) brains. These suggested that p70S6K might contribute to tau related pathologies in AD brains.

Zinc-induced anti-apoptotic effects in SH-SY5Y neuroblastoma cells via the extracellular signal-regulated kinase 1/2.

Zinc levels are increased in brain areas severely affected by Alzheimer's disease (AD) pathologies. Zinc has both protective and neurotoxic properties and can stimulate both phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways. Several kinases related to these pathways including protein kinase B (PKB), p70 S6 kinase (p70S6K), and extracellular signal-regulated kinase 1/2 (ERK1/2) are known cell survival factors and are overactivated in neurons bearing neurofibrillary tangles (NFTs) in AD. The present study aimed to determine whether anti-apoptotic effects of zinc are mediated via these signaling pathways. Zinc was used to treat SH-SY5Y neuroblastoma cells and effects investigated in relation to PKB, p70S6K, and ERK1/2 in the absence and presence of the pro-apoptotic agent staurosporine (STS). Cell damage was evaluated by measuring levels of DNA fragmentation as well as the WST-1 assay for cell viability. Results indicated that: (1) treatment with high doses of zinc (>/=400 microM) for short time periods (2 h reversed an increased DNA fragmentation due to U0126 inhibition of ERK1/2; (3) increased DNA fragmentation due to STS could be protected against by 100 microM zinc; (4) the protective effects of 100 microM zinc on STS-induced DNA fragmentation could be partially reversed by U0126. These results indicate that a zinc-induced anti-apoptotic response in SH-SY5Y cells likely occurs through ERK1/2.

Mechanism of zinc-induced phosphorylation of p70 S6 kinase and glycogen synthase kinase 3beta in SH-SY5Y neuroblastoma cells.

We have previously reported an aberrant accumulation of activated protein kinase B (PKB), glycogen synthase kinase (GSK)-3beta, extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK), p38 and p70 S6 kinase (p70S6K) in neurons bearing neurofibrillary tangles (NFTs) in Alzheimer's disease (AD). However, the mechanism by which these tau candidate kinases are involved in the regulation of p70S6K and GSK-3beta phosphorylation is unknown. In the current study, 100 microM zinc sulfate was used, and influences of various components of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways on p70S6K and GSK-3beta phosphorylation have been investigated in serum-deprived SH-SY5Y neuroblastoma cells. We found that zinc could induce an increase of phosphorylated (p) p70S6K, p-PKB, p-GSK-3beta, p-ERK1/2, p-JNK and p-p38, especially in long-term treatment (4-8 h). Treatment with different inhibitors including rapamycin, wortmannin, LY294002, and U0126, and their combinations, indicated that phosphorylation of p70S6K and GSK-3beta is regulated by rapamycin-dependent, PI3K and MAPK pathways. Furthermore, phosphorylation of p70S6K and GSK-3beta affected levels of tau unphosphorylated at the Tau-1 site and phosphorylated at the PHF-1 site, and p70S6K phosphorylation affected the total tau level. Thus, 100 microM zinc might activate PKB, GSK-3beta, ERK1/2, JNK, p38 and p70S6K, that are consequently involved in tau changes in SH-SY5Y cells.

Phosphorylated eukaryotic translation factor 4E is elevated in Alzheimer brain.

Eukaryotic translation factor 4E (eIF4E) plays a key role in regulating protein translation. It was thought that in order to maintain neuronal functions, tau protein is continuously generated to compensate those being hyperphosphorylated and compromised in its ability to promote and maintain microtubule assembly in Alzheimer's disease. If eIF4E is involved in tau mRNA translation, level of eIF4E phosphorylation should be changed. In the current study, we found a dramatic increase of phosphorylated eIF4E in Alzheimer's disease, especially in those cases with late stages of neurofibrillary changes. Level of eIF4E phosphorylation is significantly correlated with total- and Alzheimer hyperphosphorylated taus. These data suggest that the increase of eIF4E phosphorylation is involved in formation of Alzheimer neurofibrillary changes.